CN1997391A - Polyinosinic acid-polycytidylic acid-based adjuvant - Google Patents

Polyinosinic acid-polycytidylic acid-based adjuvant Download PDF

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CN1997391A
CN1997391A CNA2005800150972A CN200580015097A CN1997391A CN 1997391 A CN1997391 A CN 1997391A CN A2005800150972 A CNA2005800150972 A CN A2005800150972A CN 200580015097 A CN200580015097 A CN 200580015097A CN 1997391 A CN1997391 A CN 1997391A
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composition
antigen
dalton
polynucleotide
adjuvant
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林海祥
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Newbiomed PIKA Pte Ltd
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Abstract

The present invention provides a polynucleotide adjuvant composition and methods of use in eliciting an immune response. The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition together with an antigen (e. g. , as in a vaccine). The adjuvant compositions of the invention have particular physical properties (e. g. , molecular weight, concentration, and pH) which address the need for a safe adjuvant for eliciting an enhanced immune response. The present invention further contemplates methods of use of such adjuvant compositions, particularly in eliciting an immune response to an antigenic compound.

Description

Adjuvant based on polyinosinic acid-polycytidylicacid
Technical field
The present invention relates generally to adjunvant composition and uses these adjunvant compositions to promote immunoreactive method, and it is specific for compositions, vaccine and method in order to promotion immunogenicity of antigens (immunogenicity).It more specific about the polynucleotide adjunvant composition, contain the vaccine of this polynucleotide adjunvant composition, and utilize described polynucleotide adjunvant composition and vaccine to promote the intravital immunoreactive method of host.
Background of invention
1. the narration of correlation technique
Immune system has the specificity of generation and non-specific immunity.Generally speaking, B and T lymphocyte have the special receptor at specific antigen on their cell surface, can produce specific immunity.This immune system can be reacted with two kinds of mode specific immunes each antigen: 1) humoral immune reaction, relate to and stimulate the B cell to produce antibody or immunoglobulin, antigen presenting cell (APCs) function and helper T lymphocyte (Th1 and Th2) function, and 2) cell immune response, the generation that relate generally to the T cell, comprise cytotoxic T lymphocyte (CTLs) and other relates to ctl response for example, Th1 and/or Th2 cell and APCs.
Nonspecific immunity comprises the various kinds of cell mechanism of action; for example the phagocytosis (phagocytosis) of macrophage (macrophages) or granular cell (granulocytes) to be engulfing foreign particle or antigen, and natural killer cell (NK cell) activity etc.It is original immunologic mechanism that non-specific immunity relies on, and can not show the acquired character of specificity and Memorability, and this acquired character is the typical feature of specific immune response.Key difference between specificity and the nonspecific immunity be the former by cell mediated identification specificity antigen motif, have memory function; And the latter does not have this function.Therefore vaccination (relating to immunologic opsonin and Memorability) is that a kind of body of protecting is to resist the effective mechanism of harmful pathogen.
Adjuvant is the material of enhancing body to antigenic immunoreation or change immunoreation type, when giving jointly with antigen, it (mixes, or gives with front or rear giving this antigen) immunoreation and the immunoreactive type that can promote or change mutually for this specific antigen with this antigen.
The immunoreactive typical adjuvant of common promotion comprises: aluminium compound (this adjuvant often is commonly referred to as aluminium adjuvant " Alum "), (complete Freund's adjuvant CFA is a kind of oil in water emulsion to oil in water emulsion, contain the tubercule bacillus that drying and heat kill go out), Saponin (separates from soap matter tree QuillajaSaponoria skin, this adjuvanticity composition is called as Quile A), CpG ODN (synthesize few deoxyribonucleotide, contain CpG two polynucleotides of demethylation), MPL (deriving) from the lipopolysaccharide of Minnesota Re595 Sha Shi bacillus (Salmonella minnesota Re595), liposome (usually by making as biodegradable materials such as phospholipid) and biodegradable polymeric microspheres are (by as PLGA, multiple polymers such as poly phosphazene (polyphosphazene) and polyanhydride are made).The adjuvant character of these materials respectively has pluses and minuses through assessment.
Adjuvant is applied in vaccine for man, and particularly the child is that with the ultimate challenge of conventional vaccine most of adjuvant prescription toxigenicities and untoward reaction or adjuvant effect are not remarkable.The new technique that is used in the vaccine research and development just develops towards purification, subunit or synthetic antigen, and these antigens itself have more weak immunogenicity.Developing new adjuvant is the main challenge that the vaccine research and development are faced to improve immunogenicity/effectiveness and to lower adverse side effect.
Polynucleotide complex is on testing as multiple uses such as adjuvants.Double-stranded RNA s (dsRNAs) is the very effective bio-modification factor, can cause obvious effect by pair cell under nanomole concentration.The regulating effect of dsRNA is included in effect widely on molecule and the cellular level.
On molecular level, dsRNAs can induce as the generation of synthetic, the protein kinase of interferon (interferon), promote and organize compatible antigen and suppress biological effect such as metabolism.On cellular level, dsRNA can excite as biological effects such as pyrogen (pyrogenicity), cell division (mitogenicity), macrophage activation, activating cell immunity and initiation antiviral.The huge prospect of dsRNAs is the immunoregulation effect on the antimicrobial therapy.United States Patent (USP) the 4th, 124, No. 702 proposition dsRNAs inducing interferon in the zooblast of living produces.United States Patent (USP) the 3rd, 906, the adjuvant type vaccine antibody that No. 092 proposition contains polynucleotide or polynucleotide complex produces enhancing.People such as Houston set up PICLC (poly I: C poly-L-lysine-carboxymethyl cellulose complex) and make a kind of effective adjuvant, increase the primary antibody reaction by this and need not the auxiliary of other adjuvant (referring to Houston et al., Infection andImmunity, 14:318-9,1976C).Mould virus type dsRNA (Mycoviral dsRNA) be found can promote significantly blood coagulation antibody for the reaction of sheep red blood cell (referring to Wrightand Adler-Moore, Biochemical and Biophysical Research Communications, 131:949-45,1985).
PIC (polyinosinic acid-polycytidylicacid) can cause serious toxic reaction when being used for animal under doses, for example, people such as Phillips are reported under the 2.0mg/kg dosage the inferior chronic Canis familiaris L. PIC that gives can cause serious toxic action.The toxic voluntary activity that shows as lowers, coordinates bad, anorexia, vomiting, weight loss, the blood variation is reflected in the sinusoid capillary expansion of lobule center, hepatic necrosis, liver structural collapse and the general arthritis of the active rising of reduction hemopoietic function, alkali phosphatase and transaminase, involution of thymus, bone marrow destruction, liver (referring to Phillips et al., Toxicology and Applied Pharmacology, 18:220-30,1971).
As being studied a kind of in maximum polynucleotide complexs, PIC is ineffective when being used for monkey and human body, this be because PIC during entering primates (comprising the people) body after, understand very fast by the intravital nuclease degradation of the above animal of primates.Therefore many modes are used for improving the deficiency of PIC, for example, for high about 5 to 15 times of the comparatively former PIC of the repellence of pancreas ribonucleic acid enzyme hydrolysis, for example PIC and polyL-lysincarboxymethylcellulose complex (abbreviation PICLC) are found to be very effective antiviral or antitumor agent again by PIC and poly-L-lysine hydrobromide complex.PICLC is a kind of synthetic dsRNA that includes polynucleotide and poly-ribose cytidylic acid.Though PICLC is a kind of promising immunoregulation agent, at antimicrobial and anticancer therapy fine potential is arranged, in human trial, produce serious adverse side effect, particularly repeatedly during the high dose medication.There is the adverse side effect of report to comprise heating, hypotension, leukopenia (leucopenia), the tired pain of limbs (myalgia), thrombocytopenia (thrombocytopenia) and Polyarthralgia (polyarthralgia).Therefore this product can not be used for human body, and the toxicity of this product and stability problem must be overcome.So that making, PICLC is used safely in to use human body.The treatment effectiveness of PICLC is subject to its stability in vivo in addition.
A kind of antiviral drugs that includes polyinosinic acid-polycytidylicacid (PIC), kanamycin (kanamycin) and calcium ion (this complex is called for short Av-PICKCa) is used to treat viral infection.Confirmed that Av-PICKCa induces the generation of sending out interferon and interleukin-2 at body.Giving Av-PICKCa separately can stimulate nonspecific immune reaction as antiviral drugs, and for example Av-PICKCa directly stimulates non-generation at the specific antigen interferon down.This kind antiviral response is different from the antigen specific immune reaction that is produced very much when adjuvant and antigen give jointly.
The more important thing is that this case inventor is found to the character that Av-PICKCa has adjuvant, just when giving jointly, strengthen the ability of specific immune response with antigen.This case inventor finds that Av-PICKCa is a kind of effective adjuvant when with rabies and the common immunity of hemorrhagic fever virus antigen.
People such as Lin narration, Av-PICKCa can be used as a kind of adjuvant (referring to Lin, et al., A newimmunostimulatory complex (PICKCa) in experimental rabies:antiviraland adjuvant effects, Arch Virol, 131:307-19,1993; No. 93105862.7, Chinese patent).Chinese patent provide for No. 93105862.7 the complex that constitutes by PIC, kanamycin and calcium ion (being called for short PICKCa) people and mammal as the vaccine adjuvant purposes.
The Av-PICKCa sample is heterogeneous on molecular size and weight.In the literature, Av-PICKCa narrates with the meansigma methods or the scope of the represented sedimentation coefficient value of SF (Svedbergs) S.In one embodiment, antiviral agent Av-PICKCa is present in agent between the 5S to 8S (source A), see also Zhung J.C., Research recollectionof polyinosinic-polycytidylic acid (PIC) .The paper of fifth Chineseinterferon conference in clinical application and theory, Xian 1985, pp23-28.In other embodiments, the sedimentation coefficient of Av-PICKCa is 4S to 12S, having a meansigma methods is 6S agent (source B), perhaps sedimentation coefficient is 5S to 12S, and having a meansigma methods is 7S agent (source C), and perhaps sedimentation coefficient is 8S to 10S agent (source D), see also Hu Q.G, Tianjin Av-PICKCa ' s laboratory research and clinicalapplication, Fujian Medical Journal, 1983.12; (6): 28-30 and Hu Q.GChinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.
Utilize reduction formula mw=1,100xS2.2 is (referring to Su B.X.et al; Introduction ofBiochemical Technology, 1st Edition, Zhongshan University, 1978,356-357), the molecular weight (mw represents with dalton Daltons) that coefficient can be converted into equity falls in the Shen of the heterogeneous molecule among the Av-PICKCa.The following table demonstration is converted into daltonian result:
The characteristic of Table A: Av-PICKCa
The source Sedimentation coefficient S Molecular weight dalton
Minimum The highest On average Minimum The highest On average
A 5 8 * 38,000 107,000 *
B 4 12 6 23,000 260,000 57,000
C 5 12 7 38,000 260,000 79,000
D 8 10 * 107,000 174,000 *
*The data that do not have proposition in the list of references
People such as Lin are used for the original research as adjuvant with Av-PICKCa with a sample, the characteristic that the molecule of this sample has and the A that originates is similar, just its sedimentation coefficient is 5S to 8S, this sedimentation coefficient is to equaling the position 38,000 dalton to 107, the molecular weight of the molecule in 000 dalton's scope.(, the same) referring to Lin et al..
Believe that generally the PICKCa of form of ownership all is safe and effective on a 50-50 basis, because Av-PICKCa is a kind of form of PICKCa basically, and Av-PICKCa has been used as antiviral agent in the past.But the fact is not like this.What this case inventor was carried out studies show that, when being used as an adjuvant when the combination of PICKCa and antigen, the effectiveness of PICKCa in fact can change along with the different of molecular weight with toxicity.Can not provide best effectiveness/safety state when this case inventor finds Av-PICKCa as adjuvant, and PICKCa can cause really in some cases and makes us unacceptable adverse side effect.Therefore, industry still needs a kind of being more suitable for human use and required immunoreactive safe and effective adjuvant can be provided.The present invention can satisfy this demand and other advantage is provided, and describing in detail with reference to aftermentioned can bright Liao.
2. document
The coherent reference data is shown in down:
JP 1093540A2；
United States Patent (USP) the 4th, 124, No. 702
United States Patent (USP) the 3rd, 692, No. 899
United States Patent (USP) the 3rd, 906, No. 092
United States Patent (USP) the 4th, 389, No. 395
United States Patent (USP) the 4th, 349, No. 538
United States Patent (USP) the 4th, 024, No. 241
United States Patent (USP) the 3rd, 952, No. 097
Houston et al.，Infection and Immunity，14：318-9，1976C
Wright and Adler-Moore，Biochemical and Biophysical ResearchCommunications，131：949-45，1985
Phillips et al.，Toxicology and Applied Pharmacology，18：220-30，1971
Lin，et al.，A new immunostimulatory complex(PICKCa)inexperimental rabies：antiviral and adjuvant effects，Arch Virol，131：307-19，1993
No. the 93105862.7th, Chinese patent
Zhung J.C.，Research recollection of polyinosinic-polycytidylic acid(PIC).The paper of fifth Chinese interferon conference in clinicalapplication and theory，Siam 1985，pp23-28
Hu Q.G，Tianjin Av-PICKCa’s laboratory research and clinicalapplication，Fujian Medical Joumal，1983.12；(6)：28-30
Hu Q.G Chinese Medical and Pharmaceutical Industry Journal，1983(9)3134.
Su B.X.et al；Introduction of Biochemical Technology，1st Edition，Zhongshan University，1978，356-357
Gupta R.K.et al.，Adjuuvants-a balance between toxicity andadjuvanticity，Vaccine，11：293-306，1993
Arnon，R.(Ed.)Synthetic Vaccines 1：83-92，CRC Press，Inc.，BocaRaton，Fla.，1987
Sela，M.，Science 166：1365-1374(1969)
U.S.Pat.No.6,008,200
Ellouz et al.，Biochem.&Biophy.Res.Comm.，59：1317，1974
United States Patent (USP) the 4th, 094, No. 971
United States Patent (USP) the 4th, 101, No. 536
United States Patent (USP) the 4th, 153, No. 684
United States Patent (USP) the 4th, 235, No. 771
United States Patent (USP) the 4th, 323, No. 559
United States Patent (USP) the 4th, 327, No. 085
United States Patent (USP) the 4th, 185, No. 089
United States Patent (USP) the 4th, 082, No. 736
United States Patent (USP) the 4th, 369, No. 178
United States Patent (USP) the 4th, 314, No. 998
United States Patent (USP) the 4th, 082, No. 735
United States Patent (USP) the 4th, 186, No. 194
United States Patent (USP) the 6th, 468, No. 558
New Trends and Developments in Vaccines，edited by Voller et al.，University Park Press，Baltimore，Md.，USA，1978
Klein，J.，et al.，Immunology(2nd)，Blackwell Science Inc.，Boston(1997)
Gupa R.K.and Siber G.R.，Adjuvants for human vaccines-currentstatus，problems and future prospects，Vaccine，13(14)：1263-1276，1995
Richard T Kenney et al.Meeting Report-2nd meeting on noveladjuvants currently in/close to human clinical testing，Vaccine 202155-2163，2002
Laboratory Techniques in Rabies Edited by F X Meslin，M M Kaplan，H Koprowski 4th Edition ISBN 92 4 1544 1
Summary of the invention
The method that the invention provides a kind of polynucleotide adjunvant composition and react in order to induction of immunity.The present invention also provides a kind of immunogen constituent, comprises this polynucleotide adjunvant composition and antigen (for example these two kinds of compositions are positioned at a vaccine).Adjunvant composition of the present invention has specific physical property (for example molecular weight, concentration and pH), and these character can accord with provides a kind of effective and safe adjuvant of inducing the enhance immunity reaction and changing the immunoreation type.The present invention more provides the using method of these adjunvant compositions, particularly brings out the immunoreation for the antigen thing.
In an embodiment, the invention provides a kind of polynucleotide adjunvant composition, comprise poly-ribose inosine-poly-ribose cytidylic acid (polyriboinosinic-polyribocytidylic acid) (PIC), an antibiotic and a cation, wherein this antibiotic can be kanamycin, and this cation can be divalent ion, for example a calcium.The present invention also provides a kind of immunogen constituent, comprises this polynucleotide adjunvant composition and antigen or vaccine.
The present invention is by defining a kind of novel composition that can securely and effectively be used as an adjuvant, promoting and/or to change the intravital immunoreation of animal or human's class host, thereby promotes the lifting of knowledge.Though prior art shows that antiviral agent Av-PICKCa can be in order to as a kind of adjuvant, when the PICKCa of this kind form and antigen give jointly, only be observed and cause limited specific immune response.In addition, PICKCa is found and can causes in some cases and make us unacceptable adverse side effect.
The present invention is by providing a kind of adjunvant composition to come in response to these problems, and said composition abbreviates " PIKA " as at this, and it can be used as an adjuvant and the most effectively and safely gives animal, comprises the mankind.
PIKA is a kind of compositions, comprises a polynucleotide, an antibiotic and a cation, and said composition is become a kind of adjuvant by research and development specifically.The present invention includes several compositions, these compositionss have special product property, make these compositionss be particularly suitable for using the adjuvant of conduct in the immunogen constituent, to give the animal and/or the mankind.
Concrete going up said, the invention provides a kind of polynucleotide adjunvant composition, comprises a polynucleotide, an antibiotic and a cation, and wherein this polynucleotide can be poly-ribose inosine-poly-ribose cytidylic acid (PIC); This antibiotic is kanamycin, anthracycline (Anthracycline), butirosin sulfate (butirosin sulfate), gentamycin (gentamicin), hygromycin (hygromycin), amikacin (amikacin), dibekacin (dibekacin), nebramycin (nebramycin), beautiful its amide (metrzamide), neomycin (neomycin), puromycin (puromycin), streptomycin or streptozotocin (streptozocin); And this ion is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.
In detail, the invention provides the specification that includes a polynucleotide, an antibiotic and a cationic compositions, comprise molecular weight, concentration and pH, said composition can satisfy for the needs that cause maximum required immunoreation and safe adjuvant.
The present invention also provides a kind of immunogen constituent, comprises this polynucleotide adjunvant composition and antigen or vaccine.
In certain embodiments, the present invention presents the form of a kind of combined complete (KIT), comprises this a polynucleotide adjuvant and an immunogenic substance.
In addition, the invention provides method, by giving a host, to promote immunoreation for antigenic substance with this immunogen constituent.This host can be the mankind or animal.This gives process can be via finishing as injections such as intramuscular, intraperitoneal, intravenous or subcutaneous injections or by sucking through respiratory tract.In other embodiments, but this immunogen constituent per rectum, vagina, nose, mouth, eye, part, transdermal or Intradermal transmit.
In view of the above, the invention provides a kind of adjuvant and a kind of immunogen constituent that can be used in human body or animal body safely.
In view of the above, on the one hand, characteristics of the present invention are a kind of polynucleotide adjunvant compositions, comprise a poly-ribose inosine-poly-ribose cytidylic acid (PIC), an antibiotic and cation, wherein said composition contains on molecular weight and to be heterogeneous adjuvant molecule, and the molecular weight of this molecule is about 66,000 to 1,200,000 dalton.
In related embodiment, the polynucleotide adjunvant composition molecule in the said composition is heterogeneous on molecular weight, and wherein this molecular weight is about 300,000 to 1,200,000 dalton, or about 66,000 to 660,000 dalton, or about 300,000 to 660,000 dalton, or about 300,000 to 2,000,000 dalton, or about 300,000 to 4,000,000 dalton, or about 500,000 to 1,000,000 dalton, or about 1,000,000 to 1,500,000 dalton, or about 1,500,000 to 2,000,000 dalton, or about 2,000,000 to 2,500,000 dalton, or about 2,500,000 to 3,000,000 dalton, or about 3,000,000 to 3,500,000 dalton, or about 3,500,000 to 4,000,000 dalton, or about 4,000,000 to 4,500,000 dalton, or about 4,500,000 to 5,000,000 dalton.
In related embodiment, the polynucleotide adjunvant composition molecule in the said composition has a mean molecule quantity, and this mean molecule quantity is equal to or higher than 150,000 dalton, or be equal to or higher than 250,000 dalton, or be equal to or higher than 350,000 dalton, or be equal to or higher than 500,000 dalton, or be equal to or higher than 650,000 dalton, or be equal to or higher than 750,000 dalton, or be equal to or higher than 1,000,000 dalton, or be equal to or higher than 1,200,000 dalton, or be equal to or higher than 1,500,000 dalton, or be equal to or higher than 2,000,000 dalton.
In view of the above, in an aspect, characteristics of the present invention are a kind of polynucleotide adjunvant compositions, comprise a poly-ribose inosine-poly-ribose cytidylic acid (PIC), an antibiotic and cation, wherein said composition contains the adjuvant molecule that is heterogeneous on molecular size, and it is about 6.43S to 24.03S that coefficient unit (Svedbergs) falls in the Shen of these adjuvant molecules.
In related embodiment, polynucleotide adjunvant composition molecule in the said composition is heterogeneous on molecular size, wherein this molecular size is about 12.8S to 24.03S, or about 6.43 to 18.31S, or about 12.8 to 18.31S, or about 12.8S to 30.31S, or about 12.8S to 41.54S, about 13.5S to 18.31S, or about 13.5S to 24.03S, or about 16.14 to 22.12S, or about 22.12S to 26.6S, or about 26.6S to 30.31S, or about 30.31S to 33.55S, or about 33.55S to 36.45S, or about 36.45S to 39.1S, or about 39.1S to 41.54S, or about 41.54S to 43.83S, or about 43.83S to 45.95S.
In other related embodiment, this polynucleotide adjunvant composition has an average settlement coefficient unit (Svedbergs), and this average Shen falls coefficient and is higher than 9, or be higher than 12, or be higher than 13.5, or be higher than 15, or be higher than 16, or be higher than 17, or be higher than 18, or be higher than 19, or be higher than 20, or be higher than 21, or be higher than 22, or be higher than 25, or be higher than 30.
In a related embodiment, the antibiotic in the said composition is kanamycin, neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, puromycin, streptomycin or streptozotocin.
In another related embodiment, this adjunvant composition more comprises a kind of calcium ion.
In another related embodiment, the cation in the said composition is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.This cation can be the form of inorganic salt or organic double compound.
The calcium ion source can be for example calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.
Special related fields, the invention provides a kind of polynucleotide adjunvant composition, comprise poly-ribose inosine-poly-ribose cytidylic acid (PIC), kanamycin and calcium, wherein said composition is included in the adjuvant molecule that is heterogeneous on the molecular weight, the molecular weight of these adjuvant molecules is about 66,000 to 1,200,000 dalton.
In related embodiment, this poly-ribose inosine-poly-ribose cytidylic acid (PIC), the molecular weight of kanamycin and calcium molecule is about 300,000 to 1,200,000 dalton, or about 66,000 to 660,000 dalton, or about 300,000 to 660,000 dalton, or about 300,000 to 2,000,000 dalton, or about 300,000 to 4,000,000 dalton, or about 500,000 to 1,000,000 dalton, or about 1,000,000 to 1,500,000 dalton, or about 1,500,000 to 2,000,000 dalton, or about 2,000,000 to 2,500,000 dalton, or about 2,500,000 to 3,000,000 dalton, or about 3,000,000 to 3,500,000 dalton, or about 3,500,000 to 4,000,000 dalton, or about 4,000,000 to 4,500,000 dalton, or about 4,500,000 to 5,000,000 dalton.
In other related embodiment, the poly-ribose inosine of this adjuvant-poly-ribose cytidylic acid (PIC), kanamycin and calcium molecular composition are heterogeneous on molecular weight, these molecules have a mean molecule quantity, this mean molecule quantity is equal to or higher than 150,000 dalton, is equal to or higher than 250,000 dalton, or be equal to or higher than 350,000 dalton, or be equal to or higher than 500,000 dalton, or be equal to or higher than 650,000 dalton, or be equal to or higher than 750,000 dalton, or be equal to or higher than 1,000,000 dalton, or be equal to or higher than 1,200,000 dalton, or be equal to or higher than 1,500,000 dalton, or be equal to or higher than 1,500,000 dalton.
Special related fields, the invention provides a kind of polynucleotide adjunvant composition, comprise poly-ribose inosine-poly-ribose cytidylic acid (PIC), kanamycin and calcium, wherein said composition is included in the adjuvant molecule that is heterogeneous on the molecular size, and it is about 6.43S to 24.03S that coefficient unit (Svedbergs) falls in the Shen of these adjuvant molecules.
In related embodiment, polynucleotide adjunvant composition molecule in the said composition is heterogeneous on molecular size, wherein this molecular size is about 12.8S to 24.03S, or about 6.43 to 18.31S, or about 12.8 to 18.31S, or about 12.8S to 30.31S, or about 12.8S to 41.54S, about 13.5S to 18.31S, or about 13.5S to 24.03S, or about 16.14 to 22.12S, or about 22.12S to 26.6S, or about 26.6S to 30.31S, or about 30.31S to 33.55S, or about 33.55S to 36.45S, or about 36.45S to 39.1S, or about 39.1S to 41.54S, or about 41.54S to 43.83S, or about 43.83S to 45.95S.
In other related embodiment, this poly-ribose inosine-poly-ribose cytidylic acid (PIC), kanamycin and calcium composition have an average settlement coefficient, and this average Shen falls coefficient and is higher than 9, or be higher than 12, or be higher than 13.5, or be higher than 15, or be higher than 16, or be higher than 17, or be higher than 18, or be higher than 19, or be higher than 20, or be higher than 21, or be higher than 22, or be higher than 25, or be higher than 30.
In certain embodiments, the present invention comprises poly-ribose inosine-poly-ribose cytidylic acid (PIC) for a kind of polynucleotide adjunvant composition, kanamycin and calcium, wherein said composition is preferably and gets rid of some compositions molecule, particularly do not have on the degree of significant immunogen effect at these molecules that are excluded, the molecular weight of the compositions molecule that wherein is excluded is for approximately or be lower than 30,000 dalton, approximately or be lower than 40,000 dalton, approximately or be lower than 50,000 dalton, approximately or be lower than 60,000 dalton, approximately or be lower than 70,000 dalton, approximately or be lower than 80,000 dalton, approximately or be lower than 90,000 dalton, approximately or be lower than 100,000 dalton, approximately or be lower than 150,000 dalton, approximately or be lower than 200,000 dalton, approximately or be lower than 250,000 dalton, approximately or be lower than 300,000 dalton, approximately or be lower than 350,000 dalton, approximately or be lower than 400,000 dalton, approximately or be lower than 450,000 dalton, approximately or be lower than 500,000 dalton, approximately or be lower than 600,000 dalton, approximately or be lower than 700,000 dalton, approximately or be lower than 800,000 dalton, approximately or be lower than 900,000 dalton, approximately or be lower than 1,000,000 dalton.
In certain embodiments, the invention provides a kind of polynucleotide adjunvant composition, comprise poly-ribose inosine-poly-ribose cytidylic acid (PIC), kanamycin and calcium, wherein be preferably and get rid of some molecule of said composition, particularly do not have the immunogen effect of significance degree at these molecules that are excluded, the molecular size of the molecule that wherein is excluded is for approximately or be lower than 4.49S, approximately or be lower than 5.12S, approximately or be lower than 5.67S, approximately or be lower than 6.16S, approximately or be lower than 6.6S, approximately or be lower than 7.02S, approximately or be lower than 7.4S, approximately or be lower than 7.77S, approximately or be lower than 9.34S, approximately or be lower than 10.64S, approximately or be lower than 11.78S, approximately or be lower than 12.8S, approximately or be lower than 13.73S, approximately or be lower than 14.59S, approximately or be lower than 15.39S, approximately or be lower than 16.14S, approximately or be lower than 17.54S, approximately or be lower than 18.81S, approximately or be lower than 19.99S, approximately or be lower than 21.09S, approximately or be lower than 22.12S.
Special related fields, the invention provides a kind of antigenic immunogenic composition in order to promotion antigen thing, comprise the compositions of this polynucleotide adjuvant.
In related embodiment, this immunogen constituent comprises this polynucleotide adjuvant and antigen.
In related embodiment, the antigen source is a kind of human antigen, a kind of non-human animal's antigen, a kind of plant antigen, a kind of bacterial antigens, a kind of fungal antigen, a kind of virus antigen, a kind of parasite antigen or a kind of cancerous protuberance antigen.
In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and a rabies antigen.
In certain embodiments, antigen can be purified into from a natural origin, synthesizes or can obtain by the genetic recombination technology by solid-phase synthesis.Antigen can comprise a protein fragments, and this protein fragments contains one or more immunogen district of this molecule.Antigen also can by live, intact cell attenuation or truncate or killed or microorganism (for example complete virion) provide.
In other embodiments, antigen comprises one or more composition, other the human antigen that stems from infectious agent, plant antigen, cancerous protuberance, anaphylaxis composition and for example can cause autoimmune disease.In other embodiments, antigen comprises one or more infectious agent, stems from virus, antibacterial, mycobacteria (Mycobacterium), fungus and the parasite any.
Polynucleotide adjunvant composition of the present invention also can be used for promoting immunoreation, and this immunoreation is at the utilization antigen that dna vaccination produced.DNA sequence in order to coding for antigens in these vaccines can be " exposed ", or is accommodated in the carrier system as lipophore.
In other related embodiment, rabies antigen is to be selected from human double somatocyte vaccine (HDCV), or hamster nephrocyte deactivation purification semple type rabies vaccine (HKC-IPRV), or the rough semple type rabies vaccine of hamster nephrocyte deactivation (HKC-ICRV), or purification type Vero cell rabies (PVRV), or purification type chick-embryo cell (PCEC), or purification type duck embryo vaccine (PDEV), or hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) or the rough rabies antigen of hamster nephrocyte deactivation (HKC-ICRA).
Special related fields, the invention provides a kind of antigenic immunogen constituent in order to promotion antigen thing, comprise the polynucleotide adjunvant composition of energy inducing antigen-specific cell immune response.
Special related fields, the invention provides a kind of antigenic immunogen constituent in order to promotion antigen thing, comprise the polynucleotide adjunvant composition of energy inducing antigen-specific B cell immune response.
Special related fields, the invention provides a kind of antigenic immunogen constituent in order to promotion antigen thing, comprise energy while inducing T cell and B cell polynucleotide adjunvant composition to the antigen specific immune response.
Aspect special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of a material, comprise this polynucleotide adjunvant composition and a hamster nephrocyte deactivation purification type rabies antigen, wherein the antigenic existence of these rabies should reach a minimum flow, for example surpasses 1 iu (IU).
In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, wherein the antigenic existence of these rabies should reach a minimum flow, for example surpass 0.25 iu, surpass 0.5 iu, surpass 1.2 ius, surpass 1.4 ius, surpass 1.6 ius, surpass 1.8 ius, surpass 2.0 ius, surpass 2.2 ius, surpass 2.4 ius, surpass 2.6 ius, surpass 2.8 ius, surpass 3.0 ius, surpass 3.2 ius, surpass 3.4 ius, surpass 3.6 ius, surpass 3.8 ius, or surpass 4.0 ius.
Special related fields, the invention provides a kind of in order to promote antigenic immunogen constituent of a material, it comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, and wherein this adjuvant and rabies antigen present about 1 to 1 ratio.
In related embodiment, this immunogen constituent comprises this polynucleotide adjunvant composition and hamster nephrocyte deactivation purification type rabies antigen, and wherein this adjuvant and rabies antigen present and is lower than 1 to 10, about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 5, about 1 to 4, about 1 to 3, about 1 to 2, about 2 to 1, about 3 to 1, about 4 to 1, about 5 to 1, about 6 to 1, about 7 to 1, about 8 to 1, about 9 to 1, about 10 to 1, or greater than 10 to 1 ratio.
Special related fields, the invention provides a kind of adjunvant composition or immunogen constituent, this immunogen constituent wherein, or be comprised in the adjunvant composition of this immunogen constituent, be to present a solid or liquid form or be positioned at solution or suspension.
Special related fields, the invention provides a kind of adjunvant composition or comprise the immunogen constituent of an adjunvant composition, wherein this adjunvant composition or immunogen constituent can be frozen drying.
In related embodiment, the invention provides a kind of combined complete (KIT), comprise this adjunvant composition and antigenic substance.
Special related fields, the invention provides a kind of purposes of polynucleotide adjunvant composition, promote host's immunoreactive medicament with preparation.
Special related fields, the invention provides a kind of in order to promote immunoreactive method for the antigen thing, comprise giving the host with one in order to the antigenic immunogen constituent that promotes the antigen thing, this immunogen constituent comprises this polynucleotide adjunvant composition.
In related embodiment, the method that gives a host with this immunogen constituent can be selected from any mode in the following method, and this method includes: the outer injection of gastrointestinal tract, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, through respiratory tract suction, rectal delivery, vagina transmission, per nasal transmission, per os transmission, through eye transmission, local transmission, transdermal transmission or Intradermal transmission.
Special related fields, the invention provides a kind of in order to promote immunoreactive method for the antigen thing, comprise and give a host in order to the antigenic immunogen constituent that promotes the antigen thing one, this immunogen constituent comprises this polynucleotide adjunvant composition, and wherein this host is an animal.
Special related fields, the invention provides a kind of in order to promote immunoreactive method for the antigen thing, comprise and give a host in order to the antigenic immunogen constituent that promotes the antigen thing one, this immunogen constituent comprises this polynucleotide adjunvant composition, and wherein this host is human.
By the follow-up content and appended graphic that is described in detail, These characteristics of the present invention and other advantage will become obvious.
Description of drawings
The 1st figure shows the relative molecular weight of Av-PICKCa and PIKA sample.
The 2nd figure shows that the vaccine-induced specificity of PIKA produces the dose dependent generation of interferon gamma cytokine.
The specific embodiment
The present invention can by follow-up for some specific descriptions of the present invention and wherein included embodiment detailed content and easier quilt is understood.
Please case in this in the entire chapter content with reference to open source literature, the content of these open source literatures is merged in the application's case as a reference, to narrate the level of the technical field of the invention fully.
Before further narrating the present invention, should bright Liao the present invention can not be limited in the described specific embodiment, because these embodiment must be various.Also should bright Liao term as used in this specification only be in order to set forth specific embodiment, but not as restriction, because scope of the present invention will be defined in the appended claim.
Unless define separately, all technology as used in this specification and scientific words all with this case under have in the technical field common knowledge the personage the same meaning of general bright Liao.Just implement preferred approach of the present invention and material now and narrated, but and any method of method and materials similar described in this description or equivalence and material all can be in order to implement or test the present invention.All open source literatures of being mentioned in this description all are incorporated in this as a reference, to disclose and to illustrate method and/or material in the described open source literature.
It should be noted that as employed term in this case description and the claim unless the front and back literary composition means and bright other meaning arranged in addition, otherwise odd number shape term " a ", " and " and " the " comprise plural shape term.Therefore, for example mention that " sentence " promptly comprises the plural form of this sentence, mention that " this fragment " promptly comprises to mention one or more fragment and have the knack of equivalent of being known in the art technology personage or the like.It should be noted that in addition claim can be formulated as any alternative composition of eliminating.Therefore, this explanation be to for example " (solely) only be arranged " as using, " having only (only) " wait the removing property term when claim constitutive requirements relevant refers content or the aforementioned basis (antecedentbasis) of use " negative sense " when limiting (" negative " limitation).
Nominal definition
Before statement detailed content of the present invention, should understand several terms that are used in this description.
" adjuvant " this term that is used in herein is meant increase or changes immunoreactive any material or the mixture of substances of host for the antigen thing.Specifically say:
1. " PICKCa " this term is to censure one prevailingly by the compositions that PIC, kanamycin and calcium constituted, and compositions does not have specific physics and immunogenicity characteristic.
2. " Av-PICKCa " is meant that PICKCa is in the commercial form that is used as antiviral agent.
3. " PIKA " is meant the present composition, comprise PIC, an antibiotic (for example kanamycin) and a cation (for example calcium), wherein said PIKA is characterised in that physical characteristic (for example molecular weight of being narrated in this description, size etc.), so that PIKA can show the characteristic of an adjuvant after giving, and have compared to PICKCa being the lower adverse side effect of example (for example toxicity reduction) and compared to Av-PICKCa being the higher effectiveness intensity of example (for example inducing the immunoreation of enhancement).
" contain the PIC molecule " or " containing the PIC thing " is to censure PIC without limitation, it is optionally by compound or make up a antibiotic (for example kanamycin) in the above compositions that contains the PIC molecule and at least one or the two in the cation (for example calcium).
The composition (for example containing the PIC molecule) that is meant said composition at preceding employed hereinafter " heterogeneous " this term of adjunvant composition of the present invention is not a homogeneous on the two physical characteristic of molecular weight, size or this.
" animal " this term comprises that the mankind and all raise and wild animal and avian, and it comprises cattle, horse, milk cattle, pig, sheep, goat, Canis familiaris L., cat, rabbit, deer, ermine, chicken, duck, goose, turkey, cockfighting etc. without limitation.
" antibody " this term comprises the antigen thing binding fragment of many strains and monoclonal antibody and these antibody, comprises Fab, F (ab ') 2, Fd, Fv fragment, and these antibody and segmental strand derivant.In addition, " antibody " this term comprises the antibody of natural generation and the antibody that non-natural takes place, comprise for example mosaic type (chimeric), difunctionality type (bifunctional) and (humanized) antibody that personalizes, and relevant synthesising different structure form (isoforms).
As used in this specification, be meant can be by any material of immune system institute identification (for example being bonded to antibody or processed, with the inducing cell immunoreation) under suitable situation for " antigen thing " this term.
" antigen " is meant a kind of material, comprise the constituent that presents the vaccine form, wherein this vaccine itself comprises the antigen thing, and can comprise or can not comprise the adjuvant except PIKA, when via suitable approach (for example outside the gastrointestinal tract) when giving, this antigen can cause immunoreation such as for example forming antibody, comprises specifically in conjunction with this antigenic antibody.Antigenic two characteristics are their immunogenicity and their antigenicity, and just they cause immunoreactive ability in vivo to immunogenicity, and antigenicity is their selectivity identification capabilities that antibody had of being produced by antigen induction just.
Term such as " cellular immunization (cell-mediated immunity) " and " cell immune response (cell-mediated immune response) " is meant the immune defence power that lymphocyte provides, and for example the T lymphocyte is at the phylactic power defensive power that is provided when being injured cell.A cell immune response generally includes lymphocytic propagation.When measuring " lymphocytic propagation ", can measure the multiplication capacity of lymphocyte in response to a specific antigen.Lymphopoiesis is meant the cell proliferation of B cell, T-accessory cell or cytotoxic t-lymphocyte (CTL).
" antigen thing effective dose " is meant that the consumption of antigen thing will cause an individual specific immune response that produces at this antigen thing, and this antigen thing optionally makes up with an adjuvant.
The meaning of " increase immunoreation " this expression way or similar expression way is, compared to previous immunoreation state, immunoreation is enhanced, improves or rises, and favourable to the host, described previous immunoreation state is for example to give immunogen constituent of the present invention immunoreation state before.
Term such as " humoral immunity (humoral immunity) " and " humoral immune reaction (humoralimmune response) " is meant the immune form that produces antibody molecule in response to antigenic stimulus.
" immunoreation " this term is meant any reaction of the immune system of a vertebrates individuality for the antigen thing.Typical immunoreation includes but not limited to cell and local and systemic humoral immune reaction, for example comprise the antigenic specificity inducing action of CD8+CTLs ctl response, comprise that T-cell proliferative response and cytokine disengage the auxiliary type T-cell effect in acting on, and the B-cell immune response that comprises antibody response.
This term of this description employed " induction of immunity reaction " is to comprise immunoreactive inducing and/or potentiation (induction and/or potentiation) prevailingly.
" generation immunoreation " this term is meant stimulation, initial or cause an immunoreation.
" strengthen (potentiating) immunoreation " and be meant that an immunoreation of both having deposited is enhanced, encourages, replenishes, increases, promotes, increases or prolongs.
" poly-I:C " or terms such as " PIC " are meant a compositions that contains poly-ribose inosine and poly-ribose cytidine nucleic acid, also are referred to as polyinosinic acid-polycytidylicacid respectively.
" immune commercial weight " this term is meant, viewed immunoreation when not having the polynucleotide adjuvant, and the antigen thing adds and is enough to immune response stimulating antigen consumption when compositions of the present invention gives jointly.
" immunopotentiation amount (immunopotentiating amount) " this term is meant, viewed antibody titer and/or cell immune response when not having the polynucleotide adjuvant, when adjuvant and antigen thing give in the present composition jointly, cause antibody titer and/or cell immune response to increase required adjuvant amount.
As used in this specification, " mixing " this term comprises any method in order to the composition of forming compositions; These methods include but not limited to fusion, distribution, dissolving, emulsifying, coagulation, suspension or other method that composition part of compositions is reasonably made up.
It is medicinal acceptable that the chemicals of " pharmaceutically acceptable salt " mean this salt, and has the required pharmacologically active of parent compound.These salt comprise: (1) closes salifiable acid, and for example mineral acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid form salt jointly; Or and for example acetic acid, propanoic acid, caproic acid, cyclopentyl propionic acid, ethanedioic acid, acetone acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxy benzenes anilide) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, 1, the 2-ethionic acid, the 2-ethylenehydrinsulfonic acid, benzenesulfonic acid, the 4-chlorobenzenesulfonic acid, the 2-LOMAR PWA EINECS 246-676-2, the 4-toluenesulfonic acid, camphorsulfonic acid, glucose formic acid, 4,4 '-di-2-ethylhexylphosphine oxide-(3-hydroxyl-2-alkene-1-carboxylic acid), the 3-phenylpropionic acid, trimethylace tonitric, three grades of butyl lactic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, the common salt that forms such as organic acid such as muconic acid; Or (2) existing acid proton in parent compound is replaced just like metal ions such as alkali metal group metal ion, alkaline earth metal ion or aluminium ions, or coordination is during just like ethanolamine, diethanolamine, triethanolamine, amido butantriol (tromethamine), meglumine organic bases such as (N-methylglucamine), formed salt.
Any processing for the intravital disease of vertebrates (particularly human) contained in " treatment " this term, and comprise: (i) prevention one has the tendency of falling ill but individuality generation disease that N goes out to fall ill; (ii) contain disease, for example stop this advancing of disease; Or (iii) releive disease, for example cause this disease to disappear.
" unit dosage forms " as used in this specification this term is meant that several are isolating unit on entity, be suitable as the unit dose that is used in human and animal's individuality, each unit contains the present composition of a scheduled volume, this scheduled volume present after as calculated one be enough to and pharmacy/pharmacology on acceptable diluent, supporting agent or the common consumption that produces required effect of carrier.
The invention introduction
The present invention promotes immunoreactive compositions and method about being used in the mankind, animal or the cell culture, this immunoreation can be body fluid and/or cell immune response.Generally speaking, this constituent comprises a kind of immunogen constituent that contains an adjuvant.The existence of this adjuvant can promote or change immunoreation.Therefore, this body fluid and/or cell immune response are more effective because of the existence of this adjuvant.In addition, this adjuvant can change immunoreactive quality by the generation of several hypotypes (heterogeneous) that influence immunoglobulin and cytokine.
The key characteristic of this adjuvant is that it can induce required immunoreation level and kind and can not cause the ability of corresponding adverse side effect.Only there is at present minority to have this specific character combination for the human adjuvant that uses through approval.The safety criterion of immunogenic substances (referring in particular to vaccine) is strict and severe the execution.Therefore, the major limitation of developing a kind of successful adjuvant is to develop and a kind ofly is enough to induce a suitable immunoreactive effective product, and can not cause corresponding adverse side effect.
Preferred embodiment of the present invention is a kind of polynucleotide adjuvant, and wherein this polynucleotide is poly-ribose inosine-poly-ribose cytidylic acid (PIC).PIC is shown as a kind of effective adjuvant individually, makes us unacceptable safety speciality but can show, and unstable in human and primate body.The invention provides a kind ofly by PIC and an antibiotic and the combined compositions that forms of cation, it can promote the required immunogen characteristic of an adjuvant, and improves safety and stable speciality.
Further basis of the present invention is that the physics and the biological nature that come from the PIKA molecule of finding adjunvant composition can influence immunoreation and adverse side effect.This case the inventor be surprised to find that during researching and developing, and adjusts some characteristic of this polynucleotide adjuvant in the aftermentioned mode, and it will become effectively and/or the person becomes more or less and has toxicity more or less.Therefore, define this adjunvant composition, promptly might more correctly describe this adjunvant composition in the characteristic that provides on preferable immunogenic response and preferable safety/stable speciality from the angle of physical characteristic.
Therefore, fully added that by its chemical composition the combination of the basic physical characteristic of the molecule that constitutes this adjuvant defines at this adjuvant of the present invention that is called the PIKA adjuvant for short.Therefore, show superior immunogen character and can use safely simultaneously in the particular form of the PIKA of animal or human's class and the most suitably be defined, comprise composition, molecular weight, molecular size, concentration and pH by one or more specific speciality that normally is combination.
PIKA comprises a polynucleotide, an antibiotic and a cation usually, wherein this polynucleotide can be poly-ribose inosine-poly-ribose cytidylic acid (PIC), and this antibiotic is amido glucosides (for example kanamycin, streptomycin, safe hundred mycins (tobramycin), neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, puromycin or a streptozotocin), and this ion is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.
" amido glucosides " antibiotic is meant that this antibiotic structurally contains the amine sugar that is engaged to an amido cyclic alcohol ring (hexose core) by glycosidic bond.Amido glucosides antibiotic is to derive from multiple streptomycete (Streptomyces) and micromonospora (Micromonospora), or synthetic production.For example, kanamycin is a kind of amido glucosides antibiotic that is obtained from soil bacteria SteptomycesKanamycetics, is used for the treatment of multiple infection, particularly the infection that is caused by Glan formula negative bacteria (Gram-negative bacteria).
The PIKA compositions is in sodium chloride/phosphate buffer via the pH value that has a pH6 to pH8 at, polyinosinic acid, poly, an antibiotic and a cation source are mixed and makes.The concentration of polyinosinic acid and poly is generally 0.1 to 10mg/ml, is preferably 0.5 to 5mg/ml and be more preferred from 0.5 to, 2.5 mg/ml.Hyperchromic value (hyperchromicityvalue) should be higher than 10%, is preferably to be higher than 15% and be more preferred from and be higher than 20%.The preparation of PIC and and the combination of kanamycin and calcium preferably under the quality standard that accords with international good processing procedure standard, carry out.
In certain embodiments of the present invention, kanamycin in the polynucleotide adjunvant composition can use or be replaced by one or more antibiotic with one or more antibiotic is common, and these antibiotic are selected from and comprise in the group that safe hundred mycins, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide, neomycin, puromycin, streptomycin and streptozotocin constituted.The concentration of the antibiotic in the polynucleotide adjunvant composition of the present invention (for example kanamycin etc.) is generally about 10 to 100,000 units/ml, is preferably about 100 to 10,000 units/ml, and is more preferred from about 500 to 5,000 units/ml.
In certain embodiments of the present invention, this polynucleotide adjunvant composition more comprises a cation (cation), bivalent cation normally, and a cation of an alkali metal group metal normally.In compositions of the present invention, this cation generally is used as a cationic source, for example a kind of salt or complex, for example a kind of organic or inorganic salt or complex, and normally a kind of inorganic salt or organic double compound.Typical cation comprises but must not be limited to calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc.
Can be furnished with cation (for example calcium) in the compositions of the present invention, this cationic concentration position is preferably about 50umol to 5mmol/ml in the scope of about 10umol to 10mmol/ml, and is more preferred from about 100umol to 1mmol/ml.
As previously mentioned, cation can be any suitable salt or the form of organic double compound, comprises but must not be limited to chloride, fluoride, hydroxide, phosphate or sulfate.For example, when this cation was calcium, then this ion can be the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.
When the cation in the adjunvant composition of the present invention is calcium, then it is can be with other cation combined or replaced by other cation, these cationes comprise cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc, and wherein these ions can be the form of inorganic salt or organic double compound.
The compositions of gained further is transformed into PIKA via an extra manufacture process, and this extra manufacture process relates to separating and has the molecular size that defined and/or the molecule of weight.The similar approach of utilizing filtration, chromatography, heat treatment, centrifugalize, electrophoresis and having become standard procedure is isolated the polynucleotide molecule with particular characteristics, is to have the knack of in the art technology personage to know.
In certain embodiments of the present invention, this polynucleotide adjunvant composition further defines by the physical characteristic of molecular weight.During researching and developing, this case inventor is surprised to find that between the molecular weight of polynucleotide adjunvant composition and the effectiveness and has positive correlation.The effectiveness intensity level that the immunogen constituent that contains this polynucleotide adjunvant composition is observed comprises the ability that induction of immunity globulin and cytokine produce, and can increase along with the molecular weight of polynucleotide adjunvant composition and rise.The molecular weight of polynucleotide adjuvant can be measured by embodiment 1 described agarose gel electrophoresis.
As described in the subsequent embodiment part, this case the inventor find, and the vaccine constituent that contains the PIKA adjuvant of a diversified molecular weight can show direct association (referring to embodiment 2) between molecular weight and antigenic specificity protection effectiveness intensity.Similarly, this case the inventor find, when the combination of PIKA adjunvant composition and a rabies antigen gives the host, has directly related (referring to embodiment 3) between the molecular weight of PIKA adjunvant composition and the ability of inducing the gamma type interferon to produce.
This case inventor in 1996 during the human trial that China carried out, utilize and a kind ofly have the rabies vaccine of adjuvant and this adjuvant comprises the PICKCa of ultra high molecular weight specification, this case the inventor further identify, and resulting composition unexpectedly demonstrates has the unacceptable adverse side effect level of making us.Formerly be not disclosed the resulting result of clinical trial in 1996, be shown in now among the embodiment 4.Research for molecular weight then is shown in embodiment 5 and 6.This test is to carry out under the administration of ministry of Health of China food medication management department.Therefore, if can anticipate these adverse side effects based on knowledge at that time, then this adjuvant will can not give human experimentation in a clinical setting of being supervised.
This case the inventor find, molecular weight reaches 1.0 * 106 PIKA adjunvant composition of the present invention in preclinical test, and contain the vaccine constituent that molecular weight reaches 5.5 * 105 PIKA adjunvant composition, in specific toxicity test, demonstrate broad safety scope (referring to embodiment 7).Having highest weight and be 1.2 * 106 PIKA has successfully been used in preclinical study (referring to embodiment 3).This case inventor is carried out further studies show that safety (referring to embodiment 8) when PIKA and an antigen thing are merged into the vaccine form.
This case inventor also showed in the subsequent experimental result that China carried out in 2002, uses PIKA a kind of safe and efficient human adjuvant can be provided.This result of experiment before was not disclosed, and was presented at now among the embodiment 9.
Therefore, with the aforementioned basis that is viewed as, the molecule that is comprised in the preferred embodiment of PIKA has physical characteristic on molecular weight and/or size, these physical characteristics can increase the benefit of rendeing a service intensity and effectiveness, provide a suitable safety scope not cause any adverse side effect simultaneously.Existing molecule in Av-PICKCa, the position molecular weight ranges than low side, can be effective as a kind of antiviral synthetic, but when in an immunogen constituent, using, significantly than the molecular composition poor efficiency of PIKA as adjuvant.In addition, PIKA has shown to have the safety performance better than PICKCa.
Therefore, a molecular weight that critical aspects is present composition PIKA of the present invention.
The creativity of PIKA is formed and is generally comprised one group or a group molecule, wherein these molecules have physical characteristic on for example molecular weight and/or size, these physical characteristics provide required effect in induction of immunity reaction, and lower better or avoid adverse side effect (when for example giving PICKCa concurrent adverse side effect).Generally speaking, the molecule of PIKA is heterogeneous on molecular weight and/or size.
Except as otherwise noted, otherwise the adjunvant composition PIKA of the present invention that is generally called in this description comprises PIC, and this PIC can be mutually compound with an antibiotic (for example kanamycin) and a cation (for example calcium).Molecule in the PIKA is heterogeneous on molecular weight (for example estimating with dalton) or size (for example estimating with sedimentation coefficient).
When using a scope to describe the heterogeneous character (for example molecular weight or size) of PIKA molecule, the description of this scope be meant in this manual the PIKA molecule in compositions molecular weight or the summary of size under and the upper limit, rather than hint or point out that said composition has each molecular weight or the big or small PIKA molecule of represent in this scope.Therefore, for example molecular weight ranges is about 66,000 to 1,200,000 dalton are meant that about 66,000 dalton are to about 1,200,000 daltonian PIKA molecule is comprised in the compositions, and 88,000 daltonian PIKA molecules must not be present in the compositions (though this molecule can exist really).
When defining the physical characteristic of the PIKA molecule in the present composition with molecular weight ranges, these PIKA molecules are heterogeneous on molecular weight, and wherein this molecular weight ranges is about 300,000 to 660,000 dalton, about 300,000 to 1,200,000 dalton, about 66,000 to 660,000 dalton, or about 66,000 to 1,200,000 dalton.
The present invention also thinks and has the compositions of the PIKA molecule that is heterogeneous on molecular weight, and wherein this molecular weight ranges is about 300,000 to 2,000,000 dalton, about 300,000 to 4,000,000 dalton, about 500,000 to 1,000,000 dalton, about 1,000,000 to 1,500,000 dalton, about 1,500,000 to 2,000,000 dalton, about 2,000,000 to 2,500,000 dalton, about 2,500,000 to 3,000,000 dalton, about 3,000,000 to 3,500,000 dalton, about 3,500,000 to 4,000,000 dalton, about 4,000,000 to 4,500,000 dalton, or about 4,500,000 to 5,000,000 dalton.The position all is present in the compositions at the upper and lower limit place and position of these scopes PIKA molecule in these scopes.
When defining the physical characteristic of the PIKA molecule in the present composition with mean molecule quantity, the mean molecule quantity of this PIKA molecule can be equal to or higher than 150,000 dalton, be equal to or higher than 250,000 dalton, be equal to or higher than 350,000 dalton is equal to or higher than 500,000 dalton, be equal to or higher than 650,000 dalton, be equal to or higher than 750,000 dalton is equal to or higher than 1,000,000 dalton, be equal to or higher than 1,200,000 dalton, be equal to or higher than 1,500,000 dalton, or be equal to or higher than 2,000,000 dalton.
When defining the physical characteristic of the PIKA molecule in the present composition with sedimentation coefficient, sedimentation coefficient is a tolerance of molecular weight and size, and the Shen of this PIKA molecule falls coefficient (S) and can be and be higher than 9S or be higher than about 12S or be higher than about 13.5S, or be higher than 15S, or be higher than 16S, or be higher than 17S, or be higher than 18S, or be higher than 19S, or be higher than 20S, or be higher than 21S, or be higher than 22S, or be higher than 25S, or be higher than 30S.
In certain embodiments, the invention provides a kind of polynucleotide adjunvant composition, comprise poly-ribose inosine-poly-ribose cytidylic acid (PIC), kanamycin and calcium, wherein said composition is got rid of a molecule that can record quantity, and the molecular weight of these molecules approximately or be lower than 30,000 dalton, approximately or be lower than 40,000 dalton, approximately or be lower than 50,000 dalton, approximately or be lower than 60,000 dalton, approximately or be lower than 70,000 dalton, approximately or be lower than 80,000 dalton, approximately or be lower than 90,000 dalton, approximately or be lower than 100,000 dalton, approximately or be lower than 150,000 dalton, approximately or be lower than 200,000 dalton, approximately or be lower than 250,000 dalton, approximately or be lower than 300,000 dalton, approximately or be lower than 350,000 dalton, approximately or be lower than 400,000 dalton, approximately or be lower than 450,000 dalton, approximately or be lower than 500,000 dalton, approximately or be lower than 600,000 dalton, approximately or be lower than 700,000 dalton, approximately or be lower than 800,000 dalton, approximately or be lower than 900,000 dalton, or approximately or be lower than 1,000,000 dalton.In this embodiment, do not have at these molecules that are excluded that to get rid of these lower molecular weight molecules on the degree of significant immunogen effect be particularly advantageous.
This case inventor shows in specific toxotest, and comprising the PIKA that molecular weight reaches 1.0 * 106 daltonian molecules is safe (referring to embodiment 7) for animal.The PIKA that includes highest weight and be 1.2 * 106 daltonian molecules uses before clinical in the test (referring to embodiment 3) safely.When PIKA is used in an immunogen constituent, also show and have safety (referring to embodiment 8).The composition of PIKA provides advantage on effectiveness.Include the PIKA that molecular weight is low to moderate 6.6 * 105 daltonian molecules and when being used in the mankind and animal, also can induce effective immunoreation, and have the safety of wide wide region.The molecular weight of needed smallest molecule is increased to 6.6 * 105 dalton, and is preferably and is promoted to 3.0 * 105 dalton, can improve the effectiveness of adjuvant, and need not on safety standards, to compromise.
This case inventor finds that further the concentration of polynucleotide adjunvant composition may have influence on the molecular weight of contained molecule in the said composition.The molecular weight of PICKCa has shown that meeting increases (referring to embodiment 5) along with the rising of the concentration of adjunvant composition.This case the inventor observe, and the rising of polynucleotide adjuvant concentration can cause PICKCa molecule coalescent (or claiming coagulation), and makes molecule have higher molecular weight.This process has been shown as irreversible.Therefore, the polynucleotide adjunvant composition is follow-up is diluted in the reduction that can not cause adjuvant molecule molecular weight in the suitable medium.As observed among the embodiment 6, when the concentrated macromole form of polynucleotide adjunvant composition and rabies antigen were combined, the result obtained a compositions that remains with the high molecular scope.In the human clinical trial, demonstrate adverse side effect (referring to embodiment 4) with the formed a kind of rabies vaccine of this mode.
PIKA compositions of the present invention can be allocated in any physiologically acceptable buffer, but is good with phosphate buffer.Also can use as the replacement product of other acceptable buffer such as acetate, Tris (tris), bicarbonate, carbonate as phosphate buffer.
The pH value of aqueous solution composition is preferable between 4.0 and 10.0, but better be that pH value with this system is adjusted to 6 to 8.5, this pH value can significantly not reduce other stability of forming part, and is unlikely to incompatible on physiology.In certain embodiments, the moisture of this immunogen constituent is the normal saline of bufferingization.When desiring to give these constituents in the outer mode of gastrointestinal tract, be preferably that these solution is made tension force (being osmotic pressure) is identical with normal physiological body fluid basically, giving back tissue bulking or fast Absorption because of the diversity ion concentration between constituent and the physiological fluid to avoid constituent.
The consumption of bufferingization normal saline in these constituents is that constituent numerical value is reached the required consumption of integer.That is to say that enough buffering normal saline solution consumptions will mix mutually with other composition and reach 100%, so that constituent reaches volume required.
In certain embodiments, antigen can be purified and get, got by solid phase synthesis from a natural origin, or is obtained by gene recombination technology.Antigen can comprise a protein fragments, and this protein fragments contains one or more immunogen district of this molecule.Antigen also can from live, intact cell or microorganism (for example complete virion) attenuation or truncate or that be inactivated provide.
In other embodiments, antigen comprises one or more composition, other the human antigen that stems from infective micro-organisms, plant antigen, cancerous protuberance, anaphylaxis material and for example can cause autoimmune disease.In other embodiments, antigen comprises one or more infective micro-organisms, stems from virus, antibacterial, mycobacteria, fungus and the parasite any.
Polynucleotide adjunvant composition of the present invention also can be used for promoting immunoreation, and this immunoreation is at the utilization antigen that dna vaccination produced.DNA sequence in order to antigen encoding in these vaccines can be " exposed ", or is accommodated in the carrier system as liposome.
In certain embodiments, this polynucleotide adjunvant composition can be used in combination with vaccine.Vaccine is can contain other adjuvants or not contain other adjuvants.Included vaccine kind is infection disease, anticancer, antiallergic, anti-autoimmune and immunological contraception.
The present invention also thinks and polynucleotide adjuvant of the present invention and the antigenic applied in any combination of any suitable rabies.
In certain embodiments, rabies antigen can be as the rough rabies antigen rough rabies antigens of deactivation such as (HKC-ICRA) of hamster nephrocyte deactivation, or hamster nephrocyte deactivation purification type rabies antigen deactivation purification type rabies antigens such as (HKC-IPRA).
In certain embodiments, this polynucleotide adjunvant composition can use jointly with a rabies vaccine.The rabies vaccine that is fit to is commercial that buy or still in research and development, comprise deactivation type, subunit type, gene recombinaton type and polypeptide type vaccine, for example human double somatocyte vaccine (HDCV), or hamster nephrocyte deactivation purification semple type rabies vaccine (HKC-IPRV), or the rough rabies vaccine of hamster nephrocyte deactivation (HKC-ICRV), or purification type Vero cell rabies (PVRV), or purification type chick-embryo cell (PCEC), or purification type duck embryo vaccine (PDEV).But these rabies vaccine are not the cell immune response that can both induce before exposure and have importance after exposing in the immunization.When polynucleotide adjunvant composition (for example PIKA) and rabies vaccine give jointly, the immunoreation that is caused comprises: non--specific reaction (for example macrophage function rising), humoral response (for example promoting the generation of specific antibody) and cell immune response (for example produce cytokine, comprise interferon and interleukin-2).
In certain embodiments, the invention provides a kind of combined complete (KIT), comprise this polynucleotide adjuvant and antigen thing.
The immunogen constituent of a kind of PIKA of including can cause specific immune response in two kinds of modes: i) humoral immune reaction, (other cell also relates to the antibody response process that produces to relate to stimulation B cell generation antibody or immunoglobulin, antigen-oblatio cell (APCs for example, comprise macrophage) and helper T lymphocyte (Th1 and Th2), and ii) cell immune response, the generation that relate generally to the T cell, comprise cytotoxic T lymphocyte (CTLs) and other relates to ctl response for example, Th1 and/or Th2 cell and APCs.Industry has been known in order to the method for assessing intraindividual body fluid and/or cell immune response (referring to embodiment 10,11,12 and 13).
In addition, this polynucleotide adjunvant composition can (for IgG s be IgG1, IgG2, IgG3 and IgG4 by influencing the immunoglobulin hypotype (heterogeneous) that be produced; For mice IgGs is IgG1, IgG2a, IgG2b and IgG3) and their affinity, immunoreactive character changed.
In the Mus body, the reaction of being regulated and control by the Th1 cell can produce IgG1, IgG2a, IgG2b and less I gG3, also helps producing cell immune response at antigen.If regulated and control by Th2 type cell for antigenic IgG reaction, then it is mainly the generation of promoting IgGI and IgA.
The NIH that utilizes a kind of constituent that is made of PIKA adjuvant and hamster nephrocyte deactivation purification type rabies antigen to carry out demonstrates with rendeing a service experiment accident, and the immunity intensity of this constituent needs minimum antigenic an existence of rabies (referring to embodiment 14).The effectiveness intensity of this constituent increases sharply with respect to the antigenic existence of extra rabies that surpasses 1 IU antigen amount.Therefore, through observing, this constituent is to peak when there is the rabies antigen of the 1.5IU to 2.5IU that has an appointment in constituent rendeing a service advancing the speed on the intensity.This NIH renders a service experiment and is described in: Laboratory Techniques in Rabies, and Edited by F X Meslin, M MKaplan H Koprowski, 4th Edition, ISBN 92 4 1,544 1.
The test that utilizes a kind of constituent that is made of PIKA adjuvant and hamster nephrocyte deactivation purification type rabies antigen to carry out shows that the immunity intensity of this constituent surpasses the antigen volume along with the adjuvant volume and increases.When PIKA increased for the antigenic ratio of hamster nephrocyte deactivation purification type rabies, this effectiveness intensity can increase, and the preferred volume ratio is for being higher than 3: 1.(referring to embodiment 15)
The present invention thinks and with polynucleotide adjuvant of the present invention and the common method of using of antigen, for example to induce intravital antigenic specificity humoral response of machine and/or specific T-cells immunoreation.Inductive immunoreation can be in the individuality that does not have immunity before for antigenic reaction, or can be in order to promote existing immunoreation (for example Booster) before.
In certain embodiments, PIKA adjunvant composition and a kind of immunogen constituent that comprises PIKA adjuvant and antigen thing can be by cold Frozen dryings, and the solid form long time is stable to be preserved to be.Cold Frozen seasoning is to have the knack of the art technology personage to know.The immunogen constituent lyophilized products that will contain PIKA and antigen thing is dissolved in water and shows that it keeps original effectiveness level (referring to embodiment 16).
This immunogen constituent can be prepared to and be a kind of injectable type solution, suspension or Emulsion.The preparation of required immunogen constituent prescription is described in New Trends andDevelopments in vaccines prevailingly, edited by Voller et al., University Park Press, Baltimore, Md., USA, 1978.Immunogen constituent of the present invention can be used as following dosage form, for example forms such as capsule, solution, Emulsion, suspension or elixir come gastrointestinal tract to give, any non-activity vehicle can be used for preferred, normal saline solution for example, or the phosphate-buffered normal saline solution, or can suitably dissolve any vehicle that the employed chemical compound of the inventive method is used for the inventive method.
Immunogen constituent of the present invention can utilize the multiple method afford body of knowing.In certain embodiments, the immunogen constituent can give by the outer approach of gastrointestinal tract, injection systems such as muscle, abdominal cavity, vein, subcutaneous injection for example, or suck through respiratory tract.In other embodiments, but this immunogen constituent per rectum, vagina, nose, mouth, eye, transdermal or Intradermal part give.The antigen thing of tunica envelope can rest on the injection site and reach for two weeks when the form of giving is injection, thereby provides an antigen storage that produces continuous schedule release or pulsed release in vivo to release a little.This transmission system can be made single injection type immunogen prescription at the antigen thing that needs the reaction of multiple injection ability induction of immunity.
For example, for carry out parenteral administration with aqueous solution for, this solution where necessary should be by bufferingization suitably, and dilutes with the normal saline solution of capacity or glucose earlier so that it presents and waits.These specific aqueous solutions are specially adapted to vein and intraperitoneal gives.About this point, have the knack of the art technology personage and can know operational sterile aqueous matchmaker matchmaker thing with reference to this case disclosure.Can comprise the buffer that contains or do not contain dispersant and/or antiseptic for the typical injection Vehicle that the present invention uses, and edible oil, mineral oil, cod liver oil, Squalene (squalene) ,-, two-or triglyceride, and the mixture of these compositions.
These form required accurate consumption is along with individual different, according to individual species, age, body weight and general state, and the seriousness of the disease of being treated or preventing, infection or the patient's condition, employed predetermined substance and its form that gives etc. and change to some extent.Have the knack of after the art technology personage learns the teaching content of this description, can only utilize routine experiment and determine suitable consumption.After giving for the first time, individuality can accept one or the supplementary immunization of appropriate intervals repeatedly again.
Above said content is to narrate the present invention prevailingly.Below describe embodiment and can assist to understand the present invention.These embodiment are purpose to illustrate only, but not intention is limited for scope of the present invention.When situation becomes or be giving treatment in accordance with seasonal conditions, when thinking and the present invention's variation and equivalence replacement in form.Though this description is used specific term, these terms are the usefulness of intention for explanation, rather than to be restricted to purpose.
Embodiment
Embodiment 1. measures the molecular weight of PIKA and AV-PICKCA
This embodiment illustrates the mensuration mode of PIKA adjuvant, and makes comparisons with Av-PICKCa.
Agarose gel electrophoresis is known by haveing the knack of in the art technology personage, so this description is only narrated characteristics of the present invention.Agarose gel used in the present invention has the concentration of 1.5% agarose.Molecular marker is the 100bp dna ladder shape bands of a spectrum of 100bp to 1000bp, corresponding to 6.6 * 104 to 6.6 * 105 daltonian molecular weight ranges.The concentration of 4ul load sample is 1mg/ml.The 1st figure shows a representative graph with reference to the resultant agarose gel sample result of teaching content of this paragraph.The test of five (5) different batches shows the broad range of their molecular weight distribution.Their the molecular weight upper limit is that the position is that 2.3 * 105 dalton are in 5.28 * 105 daltonian scopes to PIKA at Av-PICKCa.
The comparison of the immune effectiveness of embodiment 2.PIKA and AV-PICKCA
This embodiment shows that highest weight is that 230,000 daltonian Av-PICKCa and highest weight are that 528,000 daltonian PIKA samples are in the difference of rendeing a service on the intensity.
With the PIKA adjuvant of three batches of different molecular weights, and a collection of have corresponding to the molecule and the hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) of the molecular weight of Av-PIKA molecular weight combined.Subsequently, make the gained constituent accept the NIH potency test.
This NIH potency test is a strictness and tests comparative study in a large number, relatively to be examined rabies vaccine and standard type rabies vaccine.Infect mice after the vaccination with a rabies virus strain alive, and measure their survival rate.The rabies vaccine of different dilute strengths is given the mice of different groups.The survival rate that is exposed between the mice group of experimental and standard type vaccine is compared, and determine effectiveness intensity (the LaboratoryTechniques in Rabies of experimental vaccine, Edited by F X Meslin, M M Kaplan H Koprowski, 4th Edition, ISBN 92 4 1,544 1).
With each combination of being checked the relative vaccine standard substance of effectiveness of vaccine of PIKA adjuvant standardized, wherein the effectiveness of vaccine standard substance is 1 by demarcation, and relative effectiveness is demarcated the multiple that the more non-combined standard substance of the effectiveness that becomes to be examined combined vaccine increase.These results of table 1 general introduction.As shown in table 1, the molecular weight of PICKCa adjuvant is higher, and it is just higher to increase the tire effectiveness of gained of rabies vaccine.
Table 1. molecular weight is renderd a service the effect of intensity for rabies vaccine
The adjuvant kind Antigen Sample # The molecular weight upper limit of adjuvant ED50 Render a service intensity (IU/ml)
PIKA HKC-IPRA 20000304 5.28×105 2.10 5.00
PIKA HKC-IPRA 20000907 4.62×105 2.00 3.98
PIKA HKC-IPRA 990202 3.96×105 1.98 3.80
Av-PIC KCa HKC-IPRA 000703 2.30×105 1.88 3.00
HKC-IPRA The vaccine standard substance 1.40 1.00
Interferon between embodiment 3.PIKA and the Av-PICKCA produces relatively
This embodiment shows, has to be limited to 230,000 daltonian Av-PICKCa samples on the molecular weight and to have to be limited to the difference on the ability that inducing interferon produces between 1,200, the 000 daltonian PIKA sample on the molecular weight.
With two batches have be limited on the molecular weight 1.2 * 106 dalton and 4.6 * 105 daltonian PIKA and a collection of have be limited to 2.3 * 105 daltonian Av-PICKCa on the molecular weight and make comparisons.
Make PIKA and Av-PICKCa compositions and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) combined.To be injected in mice under these constituent percutaneous.After two hours, measure content amount in the interferon serum of every mice.The general process of measuring interferon is to have the knack of in the art technology personage to know.In simple terms, in one 96 well culture plate, under the amount in 0.15ml/ hole by about 30,000 the L929 cells of inoculation.After three days, the degree at the bottom of cell grows to the fusion hole is diluted blood serum sample with 1: 20 to 1: 640 ratio, add in the hand-hole with serum 0.1ml/ hole.Each dilute sample is all with three multiple hole tests.With these culture plates cultivate under 37 ℃ to the next day.With the blood serum sample flush away.The generation of using stomatitis herpesvirus VSV granule to detect interferon is tired.Table 2 shows is tired by the interferon of generation that these mixture are induced.As shown in table 2, the molecular weight of PIKA sample is higher, and the interferon of being induced generation is with regard to the more.
Relation between table 2. molecular weight and interferon produce
The adjuvant kind Batch # Molecular weight HKC-IPR PIKA： HKC-IPR Interferon
The daltonian upper limit A batch of # The ratio of A What produce tires
PIKA 20010601 1.20×106 20001205 4∶1 868.6
PIKA 200009-7 4.62×105 20001205 4∶1 530.6
Av-PICK Ca 000703 2.30×105 20001205 4∶1 46.4
Embodiment vaccine for man clinical trial (having toxic side effects) in 4.1996 years
This embodiment shows that PICKCa adjuvant and the combined meeting of a vaccine produce one and make us unacceptable adverse side effect level after giving human immunity.
The purpose of this research is to assess safety and the immunoreation of a kind of rabies vaccine, it is that 11.95mg/ml and molecular mass are 69 that this rabies vaccine comprises concentration, 700 (please note that molecular mass is not equal to dalton in this example, referring to embodiment 5) the PICKCa adjuvant, and the rough rabies antigen of hamster nephrocyte deactivation (HKC-ICRA).The result of above-mentioned clinical trial and conclusion are from unexposed.
The patient of 40 participation clinical trials is divided into two 20 people's group.Each group was the 1st day, the 3rd day, the 7th day, the 14th day and the dosage injection through five 2ml of muscle acceptance in the 30th day.One winding is had the rabies antigen of PICKCa adjuvant, and another winding is had the rabies antigen of aluminium adjuvant.
From the viewpoint of safety, observe with regard to body temperature, part and general symptom each time injection back 24 hours, 48 hours and 72 hours.Following observation is:
Table 3. injection has the HKC-ICRA adverse side effect afterwards of aluminium adjuvant or PICKCa
Side effect Group Volunteer's number Number with side effect
Locality PICKCa adds HKC-ICRA 20 6
Aluminium adjuvant adds HKC-ICRAx5 20 2
General PICKCa adds HKC-ICRA 20 4
Aluminium adjuvant adds HKC-ICRAx5 20 0
The general adverse side effect comprises: heating (1 example), a rash (2 example), arthralgia (2 example), lymph node great (1 example), throat's swelling (1 example).The locality untoward reaction comprises: injection site erythrosis (6 example).
This case inventor's follow-up study is with molecular weight and the size (referring to embodiment 5 and 6) of viewed side effect owing to molecule in the adjuvant.
Relation between embodiment 5.PICKCA concentration and its molecular weight
The concentration that increases the PICKCa adjuvant that shows this embodiment causes the molecular weight of constituent to increase.
PICKCa can be made and have different concentration.Suppose when PICKCa is made into variable concentrations it is a kind of polymer composites that can multi-form existence.Experiment purpose can use laser scattering technology to reach.Laser scattering technology is widely in order to the gravimetry average molecular mass (Mw) and the radius of gyration (Rg).Instrument is commercial buys and measuring process is to have the knack of in the art technology personage to know.Table 4 demonstrates, and is relevant with its concentration by the viewed PICKCa molecular weight of laser scattering technology.
Table 4. is by the viewed molecular weight of laser scattering technology
The concentration of PICKCa (mg/ml) The weight average molecular mass
11.95 6.97×104
2.00 7.30×103
1.00 2.00×103
The preceding concentration of embodiment 6.PICKCA and the relation between the vaccine molecular weight
This embodiment shows the relation between the molecular weight of the increase of PICKCa adjuvant molecular weight and gained constituent, and this gained constituent contains PICKCa adjuvant and the rough rabies vaccine of hamster nephrocyte deactivation.
The concentration antigen that can influence in the vaccine also under a cloud before the combination of PICKCa sample.Several PICKCa samples and the rough rabies vaccine of a kind of hamster nephrocyte deactivation are combined.Experiment purpose can use laser scattering technology to reach.Laser scattering technology is widely in order to the gravimetry average molecular mass (Mw) and the radius of gyration (Rg).Instrument is commercial buys and measuring process is to have the knack of in the art technology personage to know.Table 5 shows that the preceding concentration increase of the combination of PICKCa causes the Mw of rabies vaccine to increase.
Relation before the combination of table 5.PICKCa between the Mw of concentration and rabies vaccine
The concentration of PICKCa (mg/ml) The weight average molecular mass The radius of gyration (Rg)
11.95 29.6×104 17.2×102
4.00 22.2×104 15.0×102
2.00 13.8×104 11.8×102
1.00 5.60×104 7.55×102
1.00 5.29×104 6.50×102
Embodiment 7.PIKA chronic toxicity test
This experiment shows the safety speciality of PIKA adjuvant when the restriction highest weight.
Regulation (WS1-XG-050-2000) according to the China national drug standard is carried out a chronic toxicity test.In simple terms, with the amount of 0.5 milliliter/mice, will contain 0.3 milligram and have and be limited to about 525,000 mices on the molecular weight to the about 18-22 of sodium chloride solution intravenous injection to five (5) body weight of about 1,000,000 daltonian PIKA adjuvant gram.The mice of observation through injecting 7 days, and after observing end, weigh.Its result of table 6 general introduction shows that the molecular weight of PIKA adjuvant can be up to 1.0 * 106 dalton, and does not have tangible toxicity.
Table 6.PIKA adjuvant chronic toxicity test
Batch # The upper range of molecular weight (dalton) Body weight (g) before the test of mice The tail vein injection volume Mice state when test finishes Body weight after the test of mice Suggestion
200003 04 5.25×105 18-19 0.5ml/ mice Healthy 23-26 By
200101 5.20×105 18-19 0.5ml/ Healthy 22-25 By
03 Mice
200108 16 5.20×105 18-19 0.5ml/ mice Healthy 23-25 By
200105 11 1.00×106 18-20 0.5ml/ mice Healthy 24-26 By
Embodiment 8: the toxicity research of the PIKA in the vaccine constituent
The purpose of this experiment is to confirm the safety of PIKA adjuvant.
Is that 4: 1 ratio is combined with PIKA adjuvant (molecular weight 66,000 dalton to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) with PIKA:HKC-IPRA.
To be made comparisons by vaccine constituent and a kind of commercial deactivation purification semple type rabies vaccine of buying (IPRV) that PIKA and HKC-IPRA constituted, this deactivation purification semple type rabies vaccine (IPRV) contains an aluminium adjuvant.
Give mice when the 0th day, the 3rd day, the 7th day, the 14th day and the 28th day with five doses of (5) vaccine constituents.The dosage that gives is equivalent to carry out about 300 times of adult's dosage of normal human subject rabies immunization.
The result that toxicity is observed is presented at following table 7:
Table 7: the safety that gives behind the rabies vaccine is observed
Prescription Effect The 0th day The 3rd day The 7th day The 14th day The 28th day
HKC-IPRA adds PIKA Irritated 0/20 0/20 0/20 0/20 2/20
HKC-IPRA adds PIKA Dead 0/20 0/20 0/20 0/20 0/20
HKC-IPRV (comprising aluminium adjuvant) Irritated 0/20 0/20 0/20 5/20
HKC-IPRV (comprising aluminium adjuvant) Dead 0/20 0/20 0/20 2/20 7/20
Lexical or textual analysis: (observed frequency)/(sum)
Obtaining conclusion is that the more commercial IPRV that buys of this PIKA/HKC-IPRA combination is safer.
Embodiment 9:PIKA adjuvant is in the safe handling of human body
In 2002, utilize a constituent that is constituted by PIKA constituent (molecular weight 66,000 to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) to come five (5) volunteers of immunity.Give the volunteer when the 0th day, the 3rd day, the 7th day, the 14th day and the 30th day with this vaccine constituent.
Any patient is not observed part or systemic side effect after each time vaccination.
Utilize standard NIH potency test to measure the effectiveness intensity of vaccine, the result is presented at following table 8:
Table 8: the effectiveness intensity of rabies vaccine is observed
Post-immunized day ED50 Neutralizing antibody IU/ml
0 0 0
14 ＞1.9 ＞1.84
45 2.35 5.17
The above results is pointed out vaccine constituent inducing specific immunoreation that is made of PIKA and HKC-IPRA and the generation of having induced the protectiveness neutralizing antibody.
Embodiment 10. exposes back test (cell immune response)
Exposing the back test is the most clearly proof that vaccine can be eradicated cause of disease from the host's health after infected.Therefore, it is an index of vaccine-induced cell immune response.
In after exposure, checking, a wild type strain with rabies virus is come infecting mouse, inoculate the hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) that PIKA adjuvant (molecular weight ranges from 1.65 * 105 to 1.2 * 106 dalton) are arranged with combination subsequently, or combination has the HKC-IPRA of aluminum hydroxide adjuvant (aluminium adjuvant), one commercial rabies vaccine of buying (PVRV), or phosphate buffered solution (PBS).The result demonstrates the PIKA adjuvant and has promoted survival rate, referring to table 9.
Table 9. exposes back counteracting toxic substances test
9.1 the mortality rate after the treatment
Group Predetermined 80% dead dosage Predetermined 50% dead dosage
PIKA adds HKC-IPRA 2/20 0/20
Aluminium adjuvant adds HKC-IPRA 10/20 9/20
PVRV 16/20 3/20
Matched group (PBS) 15/20 14/20
9.2 the survival rate behind the vaccine therapy
Group Predetermined 80% dead dosage Predetermined 50% dead dosage
PIKA adds HKC-IPRA 90.00％ 100.00％
Aluminium adjuvant adds HKC-IPRA 50.00％ 55.00％
PVRV 20.00％ 85.00％
Matched group (PBS) 25.00％ 30.00％
By subcutaneous injection live rabies virus institute mice infected after injection the 6th hour, the 1st day, the 2nd day and the 3rd day with vaccine therapy.
The generation of embodiment 11. antigenic specificity cell immune response gamma type interferon
The generation of gamma type interferon is the active index of cellular immunization.
In this experiment, from two bit test group patients and two matched group individualities, adopt blood sample.These aspiration patients are inoculated with the PIKA rabies vaccine, and this PIKA rabies vaccine contains PIKA (the molecular weight ranges position is 66,000 to 660,000 dalton) and hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA).Vaccination in 2.5 years before collecting blood sample.
Result among the 2nd figure demonstrates, and compared to the matched group individuality, the gamma type that two bit test group patients are produced disturbs and have significant difference.
The identical HKC-IPRA that uses cultivates jointly in the PBMCs that will separate from blood sample and the original immunity.After 1 day, with the ELISPOT method, detection specificity produces the cell quantity of gamma type interferon.Observe a dosage dependence effect.
The resulting conclusion of above-mentioned observation is:
The rabies vaccine that contains PIKA adjuvant of the present invention has the ability that specificity causes that the gamma type interferon produces, and mean can the inducing specific cell immune response.(that is to say that this reaction is at rabies antigen), and be different from nonspecific reaction.
The efficiency assay of embodiment 12:PIKA
The purpose of this experiment is to show the ability of PIKA in the generation of inducing gamma type interferon and interleukin 12 (IL-12).
In clean environment, will take from the isolating splenocyte sample of normal health mice and cultivate down in the existence of variable concentrations PIKA (the molecular weight ranges position is 66,000 to 660,000).Cultivate after three days, utilize IL-12 (p40) and gamma type interferon specific antibody, test cytokine levels in the clear liquid analytically with ELISA.Experimental result is presented at following table 10:
Table 10: the in vitro test that cytokine produces
PIKA irritaiting concentration ug/ml The IFN-gamma produces concentration pg/ml IL-12P40 produces concentration pg/ml
0 3 0
0.4 23 91
2 22 98
10 30 134
50 179 186
100 559 N/A
250 1340 N/A
The resultant conclusion of above-mentioned experiment is that PIKA induction of lymphocyte dosage dependence produces gamma type interferon and IL-12 cytokine.。
In further testing, four (4) mices are given the PIKA (the molecular weight ranges position is 66,000 to 660,000) of 500ug/ mice via lumbar injection.Use phosphate buffer as negative matched group test.Injected back 5 hours, and took out blood sample and prepare serum.Utilize IL-12 (p40) and gamma type interferon specific antibody, test the cytokine levels that detects in the serum with ELISA.Experimental result is presented at following table 11:
Table 11: the in vivo test that cytokine produces
IFN-gamma concentration pg/ml IL-12P40 concentration pg/ml
PBS
4 2
PIKA 410 40
The resultant conclusion of above-mentioned experiment is that the PIKA prompting promotes antigen presenting cell function, inducing cell immunologic function.
Embodiment 13:PIKA and the antigenic common use of deactivation purification type Vero cell rabies
The purpose of this experiment is that assessment combination has the effectiveness of the PIKA of deactivation purification type Vero cell rabies (PVRV).
The PIKA and the PVRV that will have 66,000 to 660,000 daltonian molecular weight ranges are combined, to form a kind of rabies vaccine.Use the NIH potency test to assess the effectiveness intensity of obtained vaccine constituent.The result is presented at following table 12:
Table 12: for the NIH result of the test of PIKA and deactivation purification type Vero cell rabies
The vaccine constituent Antigen Adjuvant Constituent is renderd a service intensity
PVRV 0.021 IU/ml Do not have 0.46
PVRV adds PIKA 0.021 IU/ml PIKA 3.68
The conclusion that obtains is the effectiveness intensity that PIKA can promote deactivation purification type Vero cell rabies.
Embodiment 14. antigen doses
This experiment shows the hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) need have minimum flow and PIKA adjuvant, and (molecular weight ranges is from 66,000 to 660,000 dalton) is present in jointly in the constituent, to cause specific immune response with essence enhancement level.
The PIKA adjuvant that the rabies antigen of incremental change is added quantitative 0.1mg.Detect it with NIH standard rabies vaccine potency test and render a service intensity.After the measurable original effectiveness intensity that arrives increases, and before as expection, reaching the effectiveness intensity stabilization, along with an obvious and theatrical effectiveness intensity increase (referring to table 13) is observed in the increase of antigen amount.
Table 13: the antigenic vaccine potency intensity of hamster nephrocyte deactivation purification type rabies of increment
HKC-IPRA(IU) HKC-IPRA adds 0.1mg PIKA (IU) Per unit increases the growth multiple of tiring that the antigen volume production is given birth to
0.25 0.42 -
0.51 1.43 3.88
1.01 2.73 2.60
1.40 6.07 8.56
2.07 19.78 20.46
2.91 21.38 1.90
After HKC-IPRA antigen reached an IU, observing increased a spot of HKC-IPRA, and PIKA induces vaccine potency intensity obviously to increase.
Must there be minimum antigen amount in the conclusion that obtains at significant immune response before inducing.Over-drastic antigen amount to certain a bit after, the increase of antigen amount only can the effectiveness intensity of feedback to increase slightly.
The ratio of embodiment 15. antigen adjuvants
This experiment demonstrates the best combined amount of hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA) and PIKA adjuvant (the molecular weight ranges position is 66,000 to 660,000).
Utilize various dose antigen to mix mutually, add PBS to guarantee the constant of cumulative volume with the various dose adjuvant.Utilize the NIH potency test to measure the effectiveness intensity of obtained vaccine.The result is presented at table 14.
The comprehensive consideration of result of the test points out, best vaccine combination is that PIKA drops on for antigenic ratio in 3 to 1 the scope at least.
Table 14: the rabies vaccine under various antigens and the adjuvant ratio is renderd a service intensity
Sample HKC-IP PIKA PBS Ratio ED50 Render a service strong
RA (ml) (ml) (ml) PIKA：HKC- IPRA Degree (IU/ml)
A 0.2 0.8 - 4.0 to 1 2.65 7.84
B 0.2 0.7 0.1 3.5 to 1 2.46 5.2
C 0.2 0.6 0.2 3.0 to 1 2.4 4.22
D 0.2 - 0.8 0 to 1 1.85 1.43
Standard substance na na 2.52 6.7
Embodiment 16.PIKA and combination have the lyophilization of the PIKA of rabies vaccine to preserve
This experiment shows that PIKA is stable under freeze-dried.
Freeze Drying Technique has been used in the long preservation rabies vaccine, can reach more than 3 years.Whether the rabies vaccine that this case inventor wants to test PIKA (molecular weight ranges from 66,000 to 660,000) and contain PIKA preserves favourable in lyophilization.Following constituent is used in lyophilization and preserves in the test: the PIKA that i) is not frozen, adding is in restorative lyophilization type hamster nephrocyte deactivation purification type rabies antigens (HKC-IPRA), the dry constituent of frozen type that ii) adds HKC-IPRA through recovery PIKA, iii) through the commercially available rabies vaccine of restorative lyophilization type (not adding PIKA), and the rabies vaccine standard substance that iv) are not frozen.Table 15 demonstrates, and is ideal through cryodesiccated PIKA and the rabies vaccine that contains PIKA for the long preservation rabies vaccine.
The intensity of influence render a service to(for) rabies vaccine is preserved in table 15. lyophilization
Sample ED50 Relative effectivenes intensity Neutralizing antibody IU/ml
I) by lyophilization semple type rabies vaccine that PIKA diluted 2.89 2.34 15.71
Ii) contained the PIKA rabies vaccine by the lyophilization type that PBS diluted 3.00 3.00 20.23
Iii) by the commercially available rabies vaccine of lyophilization type that PBS diluted 1.85 0.21 1.43
The rabies vaccine standard substance that iv) are not frozen 2.52 1.00 6.70
Though the present invention is described with reference to specific embodiment, should understand that these embodiment make illustrative usefulness, scope of the present invention is not limited only to this.For the personage who has general knowledge in the technical field of the invention, the concrete example of selection of the present invention is clearly.The embodiment of these selections drops in the spirit and scope of the present invention.Therefore, interest field of the present invention is to be referred by appended claim, and can be supported by aforementioned disclosure.

Claims (26)

1. polynucleotide adjunvant composition, comprise: poly-ribose inosine-poly-ribose cytidylic acid (PIC), antibiotic and cation, wherein said composition contains the polynucleotide adjunvant composition molecule that is heterogeneous in molecular weight or size, wherein this molecular weight is between about 66,000 to 1, in 200, the 000 daltonian molecular weight ranges, and should size in the molecular size scope of about 6.4S to 24.0S.
2. adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from about 300,000 to 1,200,000 dalton, or this molecular size scope is from about 12.8S to 24.0S.
3. adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from about 66,000 to 660,000 dalton, or this molecular size scope is from about 6.4S to 18.3S.
4. adjunvant composition as claimed in claim 1, wherein the molecular weight ranges of this polynucleotide adjunvant composition is from about 300,000 to 660,000 dalton, or this molecular size scope is from about 12.8S to 18.3S.
5. polynucleotide adjunvant composition, comprise: poly-ribose inosine-poly-ribose cytidylic acid (PIC), antibiotic and cation, wherein this polynucleotide adjunvant composition has and is equal to or higher than 150,000 daltonian mean molecule quantity, or have the mean molecule size that is equal to or higher than 9.3S.
6. adjunvant composition as claimed in claim 5, wherein the mean molecule quantity of this polynucleotide adjunvant composition is equal to or higher than 250,000 dalton, or this mean molecule size is equal to or higher than 11.8S.
7. adjunvant composition as claimed in claim 5, wherein the mean molecule quantity of this polynucleotide adjunvant composition is equal to or higher than 350,000 dalton, or this mean molecule size is equal to or higher than 15.3S.
8. as each described adjunvant composition in the claim 1 to 7, wherein this antibiotic is kanamycin, neomycin, anthracycline, butirosin sulfate, gentamycin, hygromycin, amikacin, dibekacin, nebramycin, beautiful its amide (metrzamide), puromycin, streptomycin or streptozotocin.
9. as each described adjunvant composition in the claim 1 to 8, wherein this cation is calcium, cadmium, lithium, magnesium, cerium, caesium, chromium, cobalt, deuterium, gallium, iodine, ferrum or zinc; And wherein this cation is the form of inorganic salt or organic double compound.
10. as each described adjunvant composition in the claim 1 to 9, wherein this cation source is calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.
11. as each described adjunvant composition in the claim 1 to 7, wherein this antibiotic is a Kanamycin Sulfate, and this cation is provided by calcium chloride.
12. in the combined complete, comprise as each described adjunvant composition and antigen thing in the claim 1 to 11.
13. an immunogenic composition comprises as each described polynucleotide adjunvant composition and antigen in the claim 1 to 12.
14. immunogenic composition as claimed in claim 13, wherein this antigen is human antigen, non-human animal's antigen, plant antigen, bacterial antigens, fungal antigen, virus antigen, parasite antigen or cancerous protuberance antigen.
15. immunogenic composition as claimed in claim 14, wherein this antigen is the rabies antigens.
16. immunogenic composition as claimed in claim 15, wherein this antigen is deactivation purification type rabies antigens.
17. immunogenic composition as claimed in claim 16, wherein the deactivation purification type rabies antigen of this hamster nephrocyte of Cun Zaiing is greater than 1 iu.
18. as each described immunogenic composition of claim 16, wherein this polynucleotide adjunvant composition and the antigenic ratio of deactivation purification type rabies of hamster nephrocyte are greater than 3 to 1.
19. as each described immunogenic composition of claim 1 to 18, wherein this immunogenic composition comprises the adjuvant that can cause enhanced special body fluid and/or cell-mediated immune responses simultaneously.
20. as claim 1 to 19 each described adjunvant composition or immunogenic composition, wherein this adjunvant composition that comprises in this immunogenic composition or this immunogenic composition be solid form or liquid form be present in solution or suspension in.
21. as claim 1 to 20 each described adjunvant composition or immunogenic composition, wherein this adjunvant composition and/or this immunogenic composition are frozen drying.
22. one kind in order to the immune response that strengthens the antagonism original method, this method comprises: will deliver medicine to the host as each described compositions in the claim 1 to 21.
23. method as claimed in claim 22, wherein this immunogenic composition can be by being selected from an administration in the group that comprises following approach, and this approach comprises the outer drug administration by injection of gastrointestinal tract, intramuscular injection, lumbar injection, intravenous injection, subcutaneous injection, through respiratory tract suction, rectally, vagina administration, nose administration, oral administration, administration through eye, topical, transdermal or intradermal administration.
24. be used for strengthening the purposes of medicine of host's immune response in preparation as each described polynucleotide adjunvant composition of claim 1 to 23.
25. as each described method of claim 1 to 24, wherein this host is human.
26. as each described method of claim 1 to 25, wherein this host is an animal.
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